Clostridium botulinum produces one of the most toxic substances known to man and presents a serious threat to human safety. The botulinum toxin is so potent that a lethal dose for an adult may be as low as 1 μg. Botulinum toxin works by blocking conduction at the neuromuscular junction and preventing the release of acetylcholine, which results in severe flaccid paralysis. Eventually, the cholinergic autonomic blockade leads to respiratory collapse and death.
There are seven different serotypes of C. botulinum and consequently seven immunologically distinct toxins. These seven toxins are identified as BoNT-A through BoNT-G and have different characteristics. BoNT-A is the most prevalent in nature and it, along with BoNT-B, is implicated in most cases of wound botulism and infant botulism. BoNT-F produces a toxin that is 60 times stronger than the toxin produced by BoNT-B.
Though botulism infections have traditionally been food-borne, wound induced, or infant related, several countries are reported to have developed botulinum toxin compositions for use as potential biological weapons. Furthermore, the prevalence and lethality of botulinum toxin give rise to potential risks by terrorist organizations. There are several unique challenges posed by such risks. First, unlike naturally occurring botulism, a biological weapon may contain a combination of the different toxins in specific amounts, so as to cause the most damage. Second, large segments of the population may become infected simultaneously. Thus, there exists a need to develop large scale botulinum antitoxin compositions directed to all of the botulinum toxins, not just those toxins most prevalent in nature.
Previous methods of manufacturing botulinum antitoxin compositions have produced unreliable results. Typically, a single animal was immunized with all seven botulinum toxins. This produced a product with variations in the levels of different botulinum titers. In particular, it was difficult to obtain a high titer response to botulinum toxins F and G through such immunization. Furthermore, previous immunization methods used alum-precipitated toxoids, which resulted in low titered antitoxins inadequate to meet the requirements of a clinically useful antitoxin produced in sufficient quantities for treating large numbers of patients.
For example, in 1991 the United States military tested a heptavalent botulinum antitoxin developed from horse serum. See U.S. Pat. No. 5,719,267. For this antitoxin, the military injected a single horse with toxoids for all seven botulinum toxins and used the resulting antitoxins. Specifically, the antitoxins were comprised of the F(ab′)2 portion of the antibody molecule. However, this method of production was unreliable because there was no control over the various concentrations of the different antitoxins produced in the horse.
Furthermore, previous methods were not suitable for large scale production of botulinum antitoxin. For example, previous methods used Cohn fractionation for isolating botulinum antibodies from plasma. The use of Cohn fractionation, however, is less effective in large scale manufacturing because large volumes of plasma must be subjected to centrifugation and the method produces antibodies with relatively low purity.
Additionally, previous methods of de-speciating equine botulinum antibodies were not particularly effective, and were quite ineffective for producing botulinum antitoxin on a large scale basis. Such methods of digestion included the use of pepsin at 37° C. over a period of 4 hours, which often resulted in incomplete digestion. Thus, long incubation periods of approximately 18-24 hours were required to achieve complete digestion under physiological conditions. To increase the rate of incubation and decrease incubation time, the incubation temperature may be increased to 70° C. However, that temperature is not compatible with achieving an active antitoxin preparation. Alternatively, a preliminary incubation of IgG at low pH, such as pH 2.8, also facilitates enzymatic digestion. However, utilizing such a low pH causes aggregation and precipitation of equine IgG.
What is needed is a reliable method of manufacturing botulinum antitoxin, preferably on a large scale basis, to produce a high titered antitoxin composition. The method of manufacture should allow for the combination of different botulinum antitoxins so that the final composition may contain high titers to all seven of the botulinum toxins.